1. Technical Field
Solid-phase peptide synthesis is a general basis of the inventive technology disclosed in the following, which more specifically relates to an apparatus employed in the isolation of synthesized peptides through cleavage from the insoluble support matrix of the solid phase, and furthermore to an associated method, facilitated by the apparatus, through which peptides produced in simultaneous multiple synthesis are together isolable, for recovery in final product form.
2. Description of the Background
Simultaneous multiple peptide synthesis is becoming of significantly increasing importance for the rapid screening or evaluation of peptides such as epitopes, agonists, antagonists or more potent structures. An eight-channel automated simultaneous solid phase peptide synthesizer has been manufactured, and has wide flexibility for syntheses. The apparatus has eight reaction vessels, each having a drainage port through which washing solvents are flushed, eight micro-syringes and an amino acid station for 160 amino acid vials. The eight independent channels allow cross-contamination-free syntheses and the generation of variable amounts of high quality peptides.
The flexibility of the automated peptide synthesizer enables the simultaneous production of peptides of different length, which can be produced according to different chemical protocols, as well as the simultaneous synthesis of different peptides. The instrument can therefore be used for the rapid evaluation of peptide synthetic chemistry or formation reaction conditions, and moreover for studies of epitopes or structure-activity relationships.
The automated peptide synthesis comprises basic amino-acid coupling cycles, wherein the carboxyl-terminal ends of peptide chains of amino acid residues coupled in synthesis are linked to styrene or other polymeric particulate beads, which can be resin coated, serving as a support matrix. The .alpha.-amino group of each amino acid added for coupling during synthesis must be protected, (i.e. blocked), so that only its activated carboxyl end is reactive (usual side groups of the amino acids are commonly protected as well). Because the compound serving as a protecting group joins to the nitrogen in the .alpha.-amino group, this may be termed "N-.alpha. protecting." The .alpha.-amino group of the terminal residue last coupled onto the growing peptide chain (or initially linked to the support matrix) is thus in a protected state and must therefore be "deprotected" prior to a subsequent coupling reaction.
Generally in the end of a coupling cycle, the elongating peptide chains are washed in solvent to cleanse them of residual coupling reagents and byproducts of the prior coupling reaction. The N-terminus away from the linked end of the peptide chain is then prepared for coupling by chemically removing the N-.alpha. protecting group; and, following another washing, a coupling reagent containing the next protected amino acid (i.e., having the N-.alpha. blocking group attached) to be joined to the deprotected N-.alpha. end of the peptidyl resin is added. Subsequent to this step, which produces amino-terminal end protected peptidyl resin intermediate in synthesis, is yet another washing in solvent, completing a normal coupling cycle in the peptide synthesis, which may be repeated to obtain a peptide product having a desired amino acid sequence.
Following the coupling cycles of a peptide synthesizing procedure in the peptide synthesizer, a final cycle ensues. Normally, after the protecting groups on the N-.alpha. ends of the peptidyl resin have been removed as prior to coupling, the synthesized, resin-linked peptides are cleansed with a final sequence of washing solvents. Alternatively, depending upon the desired application of the synthesized peptide product in final form, the peptidyl resin prior to final washing can be left in its protected state; and, given the chemistry of the protecting groups, the product can remain that way throughout the isolation process. The washed peptide product is then dried by inert gas blown through a supply opening into a reaction chamber of each reaction vessel, which contains the peptidyl resin on a filter.
In order to obtain a final peptide product, the synthesized peptides must be isolated, through cleavage From the support matrix, in a process which is carried out apart from the peptide synthesizer. This process generally requires that the peptidyl resin, i.e., the peptide in its insoluble solid phase Following synthesis, be manually transferred into a separate container, wherein it is treated with a cleaving solution which liberates the peptides from linkage to the support matrix. The resulting peptidyl solution is further isolated into final product form through a common extraction method, viz., precipitation and centrifuging.
The manually performed inter-vessel transfer of the peptidyl resin or resin-liberated peptidyl solution to containers in which the cleavage reaction or the final product extraction is carried out gives rise to mechanical loss, i.e. the tendency for some of the peptidyl resin or solution to remain behind in the vessel from which it was transferred (by pouring, for example). This consequently can reduce peptide yields, particularly in the case of simultaneous multiple peptide synthesis; moreover, the peptides are thus susceptible to contamination in the isolation process.